[CANCER RESEARCH 46, 5482-5485, November 1986] Selenium-induced Cytotoxicity of Human Leukemia Cells: Interaction with Reduced Glutathione Gerald Batist,1 Aspandiar G. Katki, Raymond W. Klecker, Jr., and Charles E. Myers Clinical Pharmacology Branch, National Cancer Institute, Bethesda, Maryland 20892 ABSTRACT Selenium exists in a number of forms with differing valence states, some of which have shown antitumor activity. We studied the tumoricidal activity of four currently available selenium forms against a human leukemia cell line and exploited the differences among them to investigate the mechanism of antitumor action. Only selenocystine and sodium selenite showed antitumor activity, and these were also the only com pounds which demonstrated significant redox chemistry, including deple tion of cellular glutathione, stimulation of glutathione reductase, and stimulation of oxygen consumption. The interaction of these two com pounds with glutathione suggests an intriguing potential role for them in cancer therapy. INTRODUCTION Selenium is an essential trace element in humans (1). Its only established function is its presence in the enzyme glutathione peroxidase (2). Using GSH2 as the reducing equivalent, this enzyme plays a significant role in detoxification of peroxides induced by oxygen radicals. As such it may be important in the toxicity of anticancer treatments that generate such reactive molecules (3). In addition, epidemiológica! and experimental data suggest an anticarcinogenic activity by selenium (4). Recent studies have also suggested an antineoplastic activity by some forms of selenium, in particular the demonstration of enhanced survival of Erlich ascites tumor-bearing mice treated with selenite (5). ¡nvitro experiments with a variety of tumor cell lines, mostly murine, have demonstrated growth inhibition by some forms of selenium (6). In fact selenocystine was used in the treatment of human leukemia in 1956 with some short- term success (7). The mechanism of this effect was thought to be competitive deprivation of cystine, but this was not proven. Others have shown that selenium compounds can cause chro mosome breaks and inhibit critical DNA synthetic enzymes (8, 9). Investigations in a variety of animal systems have suggested that there is a significant interaction of selenium with glutathi one independent of the enzyme glutathione peroxidase (10). GSH is the major non-protein sulfhydryl and serves a number of critical roles, particularly in oxidation-reduction reactions (11). The cellular concentration of GSH has recently been shown to affect significantly the efficacy and interactions of a number of anticancer drugs (12, 13). We have examined the cytotoxic effect of a number of selenium compounds on a human leukemia cell line and investigated the interaction of these compounds with intracellular GSH. MATERIALS AND METHODS Cells. The human promyelocytic leukemia cell line, HL-60, was a kind gift of Dr. T. Breitman, National Cancer Institute. HL-60 cells Received 1/8/86; revised 7/8/86; accepted 8/6/86. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1To whom requests for reprints should be addressed, at Montreal General Hospital Research Institute, Room 960, U.S.C., 1650 Cedar Avenue, Montreal, Quebec H3G 1A4, Canada. 2The abbreviations used are: GSH, reduced glutathione; PBS, phosphate buffered saline; DMF, dose modifying factor; K \„. concentration resulting in 50% growth inhibition; GSSG, oxidized glutathione; GSSeSG, selenodigluta- thione; GSSeH, selenopersulfide. were grown continuously in RPMI 1640 medium supplemented with 2 HIMglutamine, 10% calf serum (Grand Island Biological Co., Grand Island, NY), and penicillin-streptomycin (100 units/ml; 10 ml/liter), under a 5% CO2 atmosphere. The cells were subcultured twice weekly. For growth inhibition studies, 2 ml of cells at a density of 2.5 x 10s cells/ml were plated into 6-well Linbro dishes in triplicate. Viability was assessed by trypan dye exclusion, and cells were counted using a Coulter Counter on the fourth day after cells were plated and exposed to the drug. Biochemical Studies. To assess the effect of selenium on reduced glutathione and glutathione peroxidase activity, cells were incubated for 24-h periods in the highest subcytotoxic dose for selenite (5 ^M) and selenocystine (l UM),and at 100 MMfor each of selenomethionine and seténate. Reduced glutathione was measured using the fluorometric technique of Hissin and Hilf (14), which takes advantage of the fact that the fluorescent molecule ophthaldehyde selectively binds to reduced GSH at pH 8. Cells were harvested after 24 h of exposure and were washed twice in PBS and brought to a final concentration of at least 5 x 10' cells/ml in 30 nM EDTA buffer. They were sonicated three times for 10 s on ice to achieve lysis. Twenty-five % of metaphosphoric acid was added to precipitate the protein. After centrifugation, sodium phos phate buffer (NaPO4), pH 9.0 was added to the supernatant to bring the pH to 8. To 3 ml of this solution, 200 /tl of 1% ophthaldehyde dissolved in methanol was added, and the reaction was permitted to run for 15 min. Fluorescence was measured on a Perkin Elmer MPF- 44A fluorescence spectrophotometer with excitation at 350 and emis sion at 420 nm. Standards were prepared fresh daily in NaPO«buffer with 30 nM EDTA, pH 8.O. Glutathione peroxidase was measured according to the technique of Paglia and Valentine (15) using hydrogen peroxide, 0.25 HIM as sub strate. After 24 h of exposure to the various selenium compounds, the cells were washed twice in PBS and then resuspended in PBS diluted 1:1 with distilled water. After 10 min in this hypotonie solution, the cells were subjected to homogenization three times for 10 s each on ice using a Polytron homogenizer. Cell lysis was verified using phase contrast microscopy. The cell homogenates were measured in triplicate. NADPH oxidation induced by the various selenium compounds was assessed by measuring the decreasing absorban«-at 340 nm over time after addition of the selenium compound to a cuvet containing GSH (0.25 mM), NADPH (0.25 HIM),and glutathione reductase (2 units). In the absence of glutathione reductase there was no effect seen with any of the selenium compounds, indicating that there was no nonenzymatic NADPH oxidation. Oxygraphic studies were performed at 37°Cin a Gilson Model 5/6 oxygraph apparatus equipped with a Clark type electrode (Yellow Springs Instruments No. 5331). Each selenium compound to be tested was dissolved in PBS. In the absence of GSH, there was no oxygen consumption with any of the selenium compounds. Chemicals. GSH and ophthaldehyde were from Sigma Chemical Co., St. Louis, MO; glutathione reductase was from Boehringer-Mannheim, Inc., West Germany. The selenium compounds were prepared fresh for each of the studies. Sodium selenite was obtained from ICN Nutritional Biochemicals, Cleveland, OH; selenate was from ICN Pharmaceuticals, Plainsview, NY; selenomethionine from Calbiochem-Behring, La Jolla, CA; and selenocystine from Sigma. Selenocystine is the oxidized amino acid cystine containing two selenium atoms/mol of selenocystine. RESULTS Only selenocystine and sodium selenite demonstrated signif icant cytotoxic activity (Fig. 1). The IC50 for selenocystine is 5482 on July 4, 2015. © 1986 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from